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In vitro culture of bovine embryos in Menezo’s B2
´ ´
medium with or without coculture and serum: the
normalcy of pregnancies and calves resulting from
transferred embryos
John F. Hasler
)Em Tran, Inc., 197 Bossler Road, Elizabethtown, PA 17022, USA
Abstract
The present study was designed to test the efficacy, as assessed by blastocyst production, of
Ž .
culturing slaughterhouse-derived bovine oocytes in Menezo’s B2 B2 medium with or without´ ´
Ž .
serum and with or without buffalo rat cell BRL coculture. In addition, OPU-derived oocytes were cultured in B2–BRL coculture with or without serum for the first 72 h or TCM 199–BRL coculture with serum and the resulting embryos were transferred into recipients. Culture in B2 plus serum resulted in more blastocysts than B2 without serum, but both treatments produced far fewer blastocysts than did B2–BRL coculture. In addition, B2 with or without serum produced embryos of a lower stage of development, lower quality and fewer cells than did B2–BRL coculture. Serum restriction for the first 72 h of B2–BRL coculture did not result in a detectable difference in blastocyst production during 7 or 8 days of culture relative to coculture with no serum restriction. Embryos produced in B2–BRL coculture with serum restriction resulted in pregnancy rates, percentage of male fetuses, abortions, and congenital problems similar to those from B2 or TCM 199–BRL coculture with no serum restriction.q2000 Elsevier Science B.V. All
rights reserved.
Keywords: Cattle; In vitro; Embryo culture; Pregnancies; Calves
1. Introduction
Ž .
In vitro culture IVC techniques for the production of cattle embryos became widely used on a commercial basis in a number of countries during the 1990s. Although
)Fax:q1-717-653-6554.
Ž .
E-mail address: jfhasler@redrose.net J.F. Hasler .
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved.
Ž .
thousands of calves have been produced, problems with abortions, increased birth Ž weights, dystocias and higher rates of neonatal mortality have been widely reported for
. reviews, see Walker et al., 1996; Farin and Farin, 1997; Kruip and den Daas, 1997 .
Ž .
Walker et al. 1992 first described increased birth weights, lengthened gestation periods and higher neonatal mortality in sheep pregnancies resulting from in-vitro-derived pregnancies. Subsequently, the inclusion of serum during the in vitro culture of ovine
Ž
embryos was implicated in these abnormalities Thompson et al., 1995; Holm et al., .
1996 . As a result, there have been a number of recent studies directed at more clearly Ž .
defining the problem s of the inclusion of serum in the in vitro culture of both ovine Ž
and bovine embryos Popovic et al., 1997; Thompson et al., 1998; Blondin et al., 1999; Jacobsen et al., 1999; Sinclair et al., 1999; Tricoire et al., 1999; van Wagtendonk-de
.
Leeuw et al., 1999 . It remains to be demonstrated that any of the currently used in vitro systems result in the production of a statistically significant improvement in the number of ‘normal’ pregnancies. The present study was organized to determine the efficacy of the in vitro culture of bovine zygotes with and without somatic cell coculture and with or without serum for the first 72 h of culture. In addition, a survey was conducted on whether the restriction of serum during the first 72 h of development in Menezo’s B2
´ ´
ŽB2 –buffalo rat cell BRL coculture improved the normalcy of pregnancies resulting. Ž . from the transfer of embryos.2. Materials and methods
( )
2.1. Oocyte recoÕery and inÕitro maturation IVM
For Experiments 1 and 2, bovine ovaries were transported from a slaughterhouse in a plastic bag, with no additional fluid added, within an insulated box at a temperature of 26–308C. Upon arrival in the laboratory, the ovaries were rinsed with 288C tap water
Ž
and placed for 5 min into 288C tap water containing 1% Nolvasan solution Aveco, Fort
. Ž .
Dodge, IA and 1% 7=cleaning solution ICN Biochemicals, Costa Mesa, CA , after which the ovaries were rinsed copiously with tap water and maintained at ambient
Ž .
temperature 23–278C prior to aspiration. A 19-gauge 3r4-in. needle attached to a
Ž .
Pioneer Pro-Pump Pioneer Medical, Madison, CT was used to aspirate the contents of all follicles between 2 and 10 mm in diameter into a 50-ml centrifuge tube.
For Experiment 3, oocytes were aspirated from cows, primarily Holsteins, with a 5-MHz sector scanning transducer housed together in a vaginal probe with a stainless steel guide containing a 17-gauge single lumen needle. Aspiration pressure was provided by a foot-pedal-operated Pioneer Pro-Pump set at approximately 50 mm Hg. This experiment actually represents, in part, a retrospective analysis of the in vitro embryo production and transfer program provided for the owners of donor cattle housed at Em
Ž
Tran. The follicular aspirate was rinsed with PBS through an Em-Con filter Immuno .
fertiliza-Ž
tion and culture were conducted in four-well plates with no oil overlay Nunclon, .
Roskilde, Denmark . Unless stated otherwise, all chemicals were obtained from Sigma ŽSt. Louis, MO ..
Following aspiration of slaughterhouse ovaries or OPU on donor cows, oocytes were Ž
rinsed five times in a modified Tyrode’s Medium TAL-HEPES; Bio Whittaker, .
Walkersville, MD . Oocytes were placed into maturation medium within 5–9 h after the ovaries were collected at the slaughterhouse or within 30 min after OP. Groups of 30–35 experimental oocytes, or all the oocytes from individual donors were matured for 21–24 h in 0.5 ml TCM 199 with Earle’s salts, supplemented with 2.2 grl sodium bicarbonate ŽGibco BRL, Grand Island, NY , 10% heat-inactivated fetal calf serum FCS; Hyclone. Ž
. Ž
Laboratories, Logan, UT , 4mg FSH and 6 mg LH Sioux Biochemicals, Sioux Center, .
IA . The oocytes were incubated at 398C in at humidified atmosphere of 5% CO in air.2
( )
2.2. InÕitro fertilization IVF
At the end of IVM, oocytes were rinsed twice in TAL-HEPES and placed in 0.5 ml of fertilization medium, which consisted of modified tyrode–lactate–pyruvate medium ŽTALP; Bavister and Yanagimachi, 1977 . Frozen semen was thawed in a 35. 8C water
Ž .
bath and layered on a discontinuous gradient of Percoll Sigma in a 15-ml centrifuge tube. The Percoll gradient was composed of 2 ml of 90% Percoll overlaid with 2 ml of
Ž
24% Percoll, both of which were prepared with tyrode–lactate TL-HEPES; Parrish et .
al., 1988 . After 30 min of centrifugation at 700=g, the sperm pellet was recovered,
resuspended in SP-TALP and the sperm concentration determined with a hemocytome-ter. Depending on the bull, sperm were added to fertilization wells at a concentration ranging from 0.1 to 0.5=106rml. At the time, spermatozoa were added to the
fertilization wells, 20mM penicillamine, 10mM hypotaurine and 1mM epinephrine and 2 mgrml heparin were also added. After 18 h in IVF, oocytes were removed, rinsed twice in TALP, vortexed for 2 min to remove cumulus cells and then placed into in vitro culture.
2.3. IVC
2.3.1. Experiment 1
Following vortexing, oocytes aspirated from slaughterhouse-derived ovaries were
Ž . Ž
placed in 0.5 ml of one of three media: 1 B2 medium Laboratoire C.C.D., Paris,
. Ž . Ž .
France ; 2 B2 containing 10% FCS; or 3 a coculture system consisting of B2
Ž . Ž
containing 10% FCS Hyclone on a monolayer of Buffalo Rat Liver cells American .
Type Culture Collection, Rockville, MD . The BRLs were plated at a concentration of approximately 200,000, 100,000 or 50,000 cells at 24, 48 or 72 h before use. On the fourth day of IVC, the embryos were transferred to fresh media or coculture wells for all three treatments. Two replicates were conducted.
2.3.2. Experiment 2
Oocytes derived from slaughterhouse-derived ovaries were cultured in B2–BRL
Ž .
4, all oocytes were transferred to fresh coculture wells containing 10% serum. Six replicates were conducted.
2.3.3. Experiment 3
Oocytes from the donor cows were allocated randomly to B2–BRL coculture either with or without 10% serum. On Day 4 of IVC, all embryos were transferred to B2–BRL coculture including 10% serum. During the time period over which Experiment 3 was
Ž .
conducted 15 months , all embryos from an additional group of Holstein cows were
Ž .
cultured exclusively on TCM 199–BRL coculture with 10 grl BSA A-4503, Sigma and 10% FCS. In both cases, incubation was conducted at a temperature of 398C in a humidified atmosphere of 4% C0 in air. Most of the resulting embryos were transferred2
into recipients as described below.
2.4. EÕaluation of embryos
Ž
Embryos were evaluated for stage Msmorula, EBsearly blastocyst, MBs
mid-. Ž
blastocyst, XBsexpanded blastocyst, HBshatched blastocyst and grade excellent–
. Ž .
goods1, fairs2 as described previously Hasler et al., 1995 .
2.5. Staining of embryos
All blastocysts were classified for stage and grade and fixed onto slides with
Ž .
methanol–acetic acid King et al., 1979 . Following fixation, embryos were stained for 8 min in 10% giemsa, washed three times in distilled water, air-dried and coverslipped. Interphase, mitotic and pyknotic cell counts were determined by an evaluator who was
Ž .
unaware of treatment, grade or stage assignments Farin et al., 1997 .
2.6. Transfer of embryos
Embryos were removed from culture medium on Day 7 and rinsed in TALP prior to loading in a 1r4-ml straw. Single embryos were transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum of Holstein heifers that had been observed in estrus
Ž . Ž .
between 2 days before q2 and the same day as the IVF of the embryos 0 synchrony . Pregnancy was determined by rectal palpation at approximately 55–60 days of gestation. The sex of many of the pregnancies produced in this study was determined by ultrasound examination at Em Tran before the recipients were transported to the farms of the donor owners. Also, whenever possible, the sex of the resulting calves was recorded. In addition, prior to transfer, biopsies were removed from 296 embryos via
Ž .
micromanipulation and sex was determined by PCR Bredbacka et al., 1995 .
2.7. Data analysis
Table 1
Effect of B2–BRL coculture vs. cell-free B2 with or without serum on blastocyst production and the proportions of different stages of blastocysts on Day 8 of culture
Media
B2 B2qserum B2–BRL coculture
a b c
Ž . Ž . Ž . Ž .
No. blastsroocytes % 17r303 5.6 13r73 17.8 79r168 47.0
a b b
Ž . Ž . Ž . Ž .
No. EB % of total blasts 9 52.9 2 15.4 4 5.1
a a b
Ž . Ž . Ž .
No. MB 6 35.3 4 30.8 8 10.1
a b b
Ž . Ž . Ž .
No. XB 2 11.8 7 53.8 32 40.5
Ž .
No. HB 0 0 35 44.3
a,b,c
Means in rows without common superscripts differ, P-0.05.
chi-square analysis. Differences between treatment in stage, grade the number of total cells and pyknotic cells per embryo was determined by ANOVA and separate means were then analyzed by Tukey’s HSD test.
3. Results
As shown in Table 1, it is obvious from Experiment 1 that B2 medium, whether or not it was supplemented with 10% serum, did not support development of bovine zygotes to the blastocyst stage as efficaciously as did B2 supplemented with 10% serum in a BRL coculture system. Not only did coculture produce a much higher percentage of blastocysts from the oocytes that were placed into IVM, but a higher proportion of the blastocysts were more advanced in development than those in B2 either with or without serum. In addition, blastocysts resulting from culture in B2, whether or not serum was present, were composed of significantly fewer cells and were, on average, graded as
Ž .
lower in quality than blastocysts that were produced in B2–BRL coculture Table 2 . Furthermore, embryos raised with serum, either with or without coculture, were
com-Table 2
Effect of B2–BRL coculture vs. cell-free B2 with or without serum on embryo stage of development, embryo grade, percentage of pyknotic cells and total cell number
Embryo characteristics Media
B2 B2qserum B2–BRL coculture
1 a b c
Stage 2.67 3.50 4.26
a a b
Grade 2.00 1.92 1.47
a b b
% Pyknotic 26.5 14.3 8.4
a a b
No. cells 54 66 155
a,b,c
Means in rows without common superscripts differ, P-0.05. 1
Table 3
Production of bovine blastocysts from slaughterhouse-derived oocytes cultured in B2–BRL coculture with or without serum for the first 72 h of culture
Ž . Ž .
B2–BRL coculture No. oocytes No. cleaved % No. blastocysts %
Day 7 Day 8
Ž . Ž . Ž .
Serum 516 304 58.9 155 30.0 183 35.4
Ž . Ž . Ž . Ž .
No serum D1–4 504 340 67.4 162 32.1 186 36.9
posed of a lower percentage of pyknotic cells compared to embryos produced in B2 without serum.
In Experiment 2, zygotes resulting from slaughterhouse-derived oocytes and cultured
Ž .
in B2–BRL coculture without serum during Days 1–4 72 h developed into blastocysts by Days 7 and 8 at the same rate as zygotes exposed to serum throughout the culture
Ž .
period Table 3 . By Day 4 of culture, there was some sloughing of BRL cells from the bottom of the culture wells. However, this was not accompanied by any observable influence on the percentage of oocyte cleavage or the rate of embryo development in this treatment.
In Experiment 3, there was no difference among the three culture systems in the efficiency, which ranged from 17.8% to 19.6%, of embryo production from the oocytes
Ž .
that were placed into IVM Table 4 . Also, there was no difference in 60-day pregnancy rates in recipient heifers resulting from the transfer of embryos produced in the three different culture systems. The sex of embryos, as determined by three different methods Žas described in Section 2 , was not different for embryos produced among the three.
Ž .
different culture systems Table 4 . The percentage of males for each separate treatment
Ž .
and for all treatments combined 54.2% did not differ significantly from a sex ratio of 50:50. There were no differences in the distribution of embryo stages produced in
Ž .
B2–BRL coculture with or without serum Table 5 . However, B2 cocultures, either
Table 4
Embryo production and pregnancy rate following transfer of embryos derived from OPU-collected oocytes cultured in B2–BRL coculture with or without serum for the first 72 h of culture or TCM 199–BRL coculture with serum
B2 TCM 199
Serum No serum Serum
No. oocytes 3250 3002 1725
No. embryos 638 535 313
% Embryosroocytes 19.6 17.8 18.1
a
No. transfers 467 382 129
Ž . Ž . Ž . Ž .
No. pregnant % 223 47.8 180 47.1 65 50.4
b
Ž . Ž . Ž . Ž .
No. males % 186r345 53.9 152r280 54.3 36r65 55.4
a
Does not include embryos that were biopsied or frozen. b
Table 5
Distribution of different stages of embryos derived from OPU-collected oocytes cultured in B2–BRL coculture with or without serum for the first 72 h of culture or TCM 199–BRL coculture with serum
Ž .
No. embryos % total B2 TCM 199
Serum No serum Serum
a b
Ž . Ž . Ž .
Morulae 84 13.2 52 9.7 27 8.6
Ž . Ž . Ž .
Early blastocysts 186 27.2 149 27.8 95 30.4
a a b
Ž . Ž . Ž .
Mid blastocysts 191 29.9 183 34.2 139 44.4
a a b
Ž . Ž . Ž .
Expanded blastocysts 155 24.3 137 25.6 51 16.3
a a b
Ž . Ž . Ž .
Hatched blastocysts 22 3.4 14 2.7 1 0.3
Total 638 535 313
a,b
Means in rows without common superscripts differ, P-0.05.
with or without serum, produced a higher percentage of expanded and hatched blasto-cysts than did the TCM 199–BRL coculture system.
There was no difference in the percentage of spontaneous abortions, live births or
Ž .
dystocias among the three different coculture systems Table 6 . There was no way to accurately differentiate between Caesarean deliveries that were scheduled prior to the onset of labor vs. those that were performed as the result of calving problems in recipients. Nevertheless, there was no difference in the survival rate of calves following Caesarean delivery among the three coculture systems. There was, however, a
signifi-Ž .
cantly lower survival rate P-0.05 following natural delivery of all calves combined
Ž .
for the three coculture systems 187r228s82.0% compared to the total of all calves
Ž .
that survived Caesarean delivery 55r59s93.2% . There were congenital abnormalities in calves and cases of hydro-allantois observed in recipients that received embryos from either of the two B2 coculture systems, but neither problem was observed in the
Table 6
Pregnancy and calving characteristics following transfer of embryos derived from OPU-collected oocytes cultured in B2–BRL coculture with or without serum for the first 72 h of culture or TCM 199–BRL coculture with serum
B2 TCM 199
Serum No serum Serum
a
Total no. pregnancies 273 201 65
Ž . Ž . Ž . Ž .
No. congenital malformations % 4r139 2.9 2r94 2.1 0
Ž . Ž . Ž .
No. hydro-allantois % 2r273 0.7 1r201 0.5 0
a
pregnancies resulting from embryos cultured in TCM 199. A frequently reported characteristic of the pregnancies resulting from all three culture systems was that labor was not clearly pronounced in the recipients.
4. Discussion
Ž .
Pinyopummintr and Bavister 1991 showed that although TCM 199 supported bovine blastocyst development without serum, more blastocysts and higher cell numbers were achieved when serum was included. Similarly, in the present study, although the B2 medium contained BSA, the addition of serum resulted in more blastocysts and a higher percentage of advanced stages of blastocysts. However, B2 either with or without the inclusion of serum produced significantly fewer blastocysts than did the B2 combined with BRL cells in a coculture system. It has been demonstrated previously that various coculture systems based on complex media combining any of a number of different somatic cells, including oviductal cells, cumulusrgranulosa cells, uterine and skin cells, Vero cells and BRL cells, supported a high rate of development of bovine
Ž .
embryos to blastocysts for review, see Gordon, 1994 . The percentage of early embryos Ž
that developed to blastocysts was similar for a variety of coculture cell types Aoyagi et .
al., 1990; Jiang et al., 1991; Goto et al., 1992; Pegoraro et at., 1998 . Also, it has been
Ž .
shown that when either B2 Thibodeaux et al., 1992; Xu et al., 1992 or TCM 199 ŽAoyagi et al., 1990; Jiang et al., 1991; Goto et al., 1994; Pavasuthipaisit et al., 1994;
.
Rehman et al., 1994 were combined with somatic cells in a coculture system, both media supported a higher rate of development to blastocysts than did control media without cells. In the present study, embryos cultured in a B2–BRL coculture system exhibited faster development, with a higher total number of blastocysts and higher embryo cell counts, than did B2 medium without coculture.
Coculture systems for mammalian embryos normally contain serum, as much for the benefit of the coculture cells as for possible benefits to the embryos. Because serum has
Ž
been implicated in causing heavier birth weights of in-vitro-produced lambs Walker et .
al., 1992; Thompson et al., 1995 , coculture of bovine embryos without serum was
Ž .
investigated in the present study. Farin and James 1996 previously showed that bovine embryos could be successfully cultured in TCM 199 with cumulus cell coculture without serum for the first 72 h. They reported that serum restriction did not affect the percent of embryos reaching compact morula or blastocyst stages, but that the morphological quality was on average lower than for embryos exposed to serum throughout culture. In the present study, serum restriction for the first 72 h of culture in B2 did not affect the percentage of embryos reaching blastocysts nor the distribution of different stages of blastocysts ranging from early to hatched. On a more practical basis, serum restriction did not influence the pregnancy rate. Unfortunately, it also did not decrease the incidence of abortions, dystocias or congenital problems.
Although the sex ratio of calves did not deviate from 50:50 for any of the three coculture systems in this study because of the small sample sizes, the percentage of bull
Ž .
calves for all systems combined 54.2% is virtually identical to the 54.3% males
Ž .
males following transfer of in vitro-derived embryos also has been reported variously as
Ž . Ž .
55.5% van Wagtendonk-de Leeuw et al., 1998 , 60% Reichenbach et al., 1992 , 62% ŽGuyader-Joly et al., 1993; Massip et al., 1995 , and 57.6% Agca et al., 1998 . A. Ž . number of reports showed that male bovine embryos produced in vitro grow more
Ž
rapidly than female embryos during the first 7–8 days of culture Avery et al., 1991; Xu .
et al., 1992; Carvalho et al., 1996; Tocharus et al., 1997 . Therefore, probably more male embryos are transferred because bovine IVC embryos are usually transferred on Day 7 and the most advanced embryos are usually selected for transfer.
Ž .
Kruip and den Daas 1997 analyzed information from 30 data sets on transfers of bovine IVF embryos obtained worldwide and reported that abortion rates were higher, gestation lengths and birth weights were increased, and there were higher incidences of dystocia relative to pregnancies produced by artificial insemination or embryo transfer of in vivo embryos. Specifically, the problems with pregnancies and calvings described
Ž .
previously Hasler, 1998 and in the present study closely parallel those described by
Ž .
Van Wagtendonk-de Leeuw et al. 1998 for bovine pregnancies produced by IVF embryos cultured in TCM 199 in a BRL monolayer system. Using synthetic oviduct
Ž . Ž .
fluid SOF in a non-coculture system, Thompson et al. 1998 reported that bovine blastocyst development was accelerated when serum or charcoal-treated serum was added on Day 5 compared to media containing only BSA throughout culture. Transfer of embryos following culture resulted in similar pregnancy and embryo survival rates for the three treatments and no birth weight differences were detected. Two other recent studies also failed to demonstrate any difference in birth weights or calving ease
Ž
following transfer of embryos cultured in SOF with or without serum Jacobsen et al., .
1999; Tricoire et al., 1999 . In both of these studies, the small numbers of pregnancies
Ž .
produced 5–16 per treatment may have been a factor in the failure to demonstrate a significant difference between treatments.
Ž .
Recently, using sheep as a model, Sinclair et al. 1999 compared pregnancies resulting from the transfer of in-vivo-derived embryos vs. those cultured in TCM 199 with granulosa cell coculture or in SOF with or without human serum. Fetuses were significantly heavier and growth coefficients for liver and heart for fetuses were greater from cocultured and serum-supplemented embryos compared to control embryos. The allometric coefficients for liver and heart from fetuses derived from embryos cultured in SOF without serum were not different from controls, however. Complementing this research, Holland Genetics has recently reported that the birth weight of calves produced from SOF without serum was lighter than that of calves produced from TCM
199-cocul-Ž .
ture with serum van Wagtendonk-de Leeuw et al., 2000 . These findings strongly suggest that culture of in-vitro-derived bovine and ovine embryos in a semi-defined medium without serum or coculture supplementation may result in an improvement in the normalcy of pregnancies.
Acknowledgements
acknowledged. This research was financially supported in part by Holland Genetics, Arnhem, The Netherlands.
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